The present invention relates to the use of cyanovirins to inhibit prophylactically or therapeutically influenza viral infection, as well as glycosylation-resistant cyanovirins and related conjugates, compositions, nucleic acids, vectors, host cells and methods of production and use.
The field of viral therapeutics has developed in response to the need for agents effective against retroviruses, especially HIV. There are many ways in which an agent can exhibit anti-retroviral activity (e.g., see DeClercq, Adv. Virus Res. 42, 1-55, 1993; DeClercq, J. Acquir. Imun. Def. Synd. 4, 207-218, 1991; and Mitsuya et al., Science 249, 1533-1544, 1990). Nucleoside derivatives, such as AZT, which inhibit the viral reverse transcriptase, were among the first clinically active agents available commercially for anti-HIV therapy. Although very useful in some patients, the utility of AZT and related compounds is limited by toxicity and insufficient therapeutic indices for fully adequate therapy. Also, given the subsequent revelations about the true dynamics of HIV infection (Coffin, Science 267, 483-489, 1995; and Cohen, Science 267, 179, 1995), it has become increasingly apparent that agents acting as early as possible in the viral replicative cycle are needed to inhibit infection of newly produced, uninfected immune cells generated in the body in response to the virus-induced killing of infected cells. Also, it is essential to neutralize or inhibit new infectious virus produced by infected cells.
Infection of people by influenza viruses is also a major cause of pandemic illness, morbidity and mortality worldwide. The adverse economic consequences, as well as human suffering, are enormous. Available treatments for established infection by this virus are either minimally effective or ineffective; these treatments employ amantatadine, rimantadine and neuraminidase inhibitors. Of these drugs, only the neuraminidase inhibitors are substantially active against multiple strains of influenza virus that commonly infect humans, yet these drugs still have limited utility or efficacy against pandemic disease.
Currently, the only effective preventative treatment against influenza viral infection is vaccination. However, this, like the drug treatments, is severely limited by the propensity of influenza viruses to mutate rapidly by genetic exchange, resulting in the emergence of highly resistant viral strains that rapidly infect and spread throughout susceptible populations. In fact, a vaccination strategy is only effective from year-to-year if the potential pandemic strains can be identified or predicted, and corresponding vaccines prepared and administered early enough that the year""s potential pandemic can be aborted or attenuated. Thus, new preventative and therapeutic interventions and agents are urgently needed to combat influenza viruses.
New agents with broad anti-influenza virus activity against diverse strains, clinical isolates and subtypes of influenza virus would be highly useful, since such agents would most likely remain active against the mutating virus. The two major types of influenza virus that infect humans are influenza A and B, both of which cause severe acute illness that may include both respiratory and gastrointestinal distress, as well as other serious pathological sequellae. An agent that has anti-influenza virus activity against diverse strains and isolates of both influenza A and B, including recent clinical isolates thereof, would be particularly advantageous for use in prevention or treatment of hosts susceptible to influenza virus infection.
The predominant mode of transmission of influenza viral infection is respiratory, i.e., transmission via inhalation of virus-laden aerosolized particles generated through coughing, sneezing, breathing, etc., of an influenza-infected individual. Transmission of infectious influenza virions may also occur through contact (e.g., through inadvertent hand-to-mouth contact, kissing, touching, etc.) with saliva or other bodily secretions of an infected individual. Thus, the primary first points of contact of infectious influenza virions within a susceptible individual are the mucosal surfaces within the oropharyngeal mucosa, and the mucosal surfaces within the upper and lower respiratory tracts. Not only do these sites comprise first points of virus contact for initial infection of an individual, they are also the primary sites for production and exit (e.g., by coughing, sneezing, salivary transmission, etc.) of bodily fluids containing infectious influenza viral particles. Therefore, availability of a highly potent anti-influenza virus agent, having broad-spectrum activity against diverse strains and isolates of influenza viruses A and B, which could be applied or delivered topically to the aforementioned mucosal sites of contact and infection and transmission of infectious influenza viruses, would be highly advantageous for therapeutic and preventative inhibition of influenza viral infection, either in susceptible uninfected or infected hosts.
In this regard, new classes of antiviral agents, to be used alone or in combination existing antiviral agents, are needed for effective antiviral therapy. New agents are also important for the prophylactic inhibition of viral infection. In both areas of need, the ideal new agent(s) would act as early as possible in the viral life cycle; be as virus-specific as possible (i.e., attack a molecular target specific to the virus but not the host); render the intact virus noninfectious; prevent the death or dysfunction of virus-infected cells; prevent further production of virus from infected cells; prevent spread of virus infection to uninfected cells; be highly potent and active against the broadest possible range of strains and isolates of a given virus; be resistant to degradation under physiological and rigorous environmental conditions; and be readily and inexpensively produced.
Accordingly, it is an object of the present invention to provide a method of inhibiting prophylactically or therapeutically a viral infection, specifically an influenza viral infection, of a host. It is another object of the present invention to provide glycosylation-resistant cyanovirins and related conjugates, nucleic acids, vectors, host cells and methods of production and use. These and other objects and advantages of the present invention, as well as additional inventive features, will become apparent from the description provided herein.
The present invention provides, among other things, an isolated and purified nucleic acid molecule that encodes a protein or peptide comprising at least nine contiguous amino acids of SEQ ID NO:2, wherein the at least nine contiguous amino acids comprise amino acids 30-32 of SEQ ID NO: 2 which have been rendered glycosylation resistant and wherein the at least nine contiguous amino acids have antiviral activity. Further provided is a vector comprising such an isolated and purified nucleic acid molecule and a host cell or organism comprising the vector.
Accordingly, the present invention also provides a method of producing an antiviral protein or antiviral peptide, which method comprises expressing the vector in a host cell or organism. Thus, an antiviral protein or antiviral peptide comprising at least nine contiguous amino acids of SEQ ID NO:2, wherein the at least nine contiguous amino acids comprise amino acids 30-32 of SEQ ID NO: 2 which have been rendered glycosylation resistant and wherein the at least nine contiguous amino acids have antiviral activity, is also provided as is a conjugate comprising the antiviral protein or antiviral peptide and at least one effector component selected from the group consisting of polyethylene glycol, albumin, dextran, a toxin and an immunological reagent. Compositions comprising an effective amount of the antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate are also provided.
The present invention further provides a method of inhibiting prophylactically or therapeutically a viral infection, specifically an influenza viral infection, of a host. The method comprises administering to the host an effective amount of an antiviral protein, antiviral peptide, antiviral protein conjugate or antiviral peptide conjugate comprising at least nine contiguous amino acids of SEQ ID NO:2, wherein the at least nine contiguous amino acids are nonglycosylated and have antiviral activity, whereupon the viral infection is inhibited.